This proposal describes a five-year plan for training Jun Wu to achieve her goal to become an independent investigator in metabolic research. The training and career development plan includes a compelling research project with potential clinical applications, training in laboratory techniques and didactic scientific and career development seminars and courses. The applicant has more than a decade of experiences working in molecular and cellular biology, mouse genetics and physiology. Her previous important findings have been published in many high-impact journals, and have been then cited more than 2,000 times in the subsequent works of her peers. Dr. Bruce Spiegelman, a well-recognized leader in the field of diabetes and obesity will mentor the applicant's scientific and career development. Dr. Spiegelman has trained numerous postdoctoral fellows who now have faculty positions in academic institutions. In addition, an advisory committee of highly- regarded experts in the field of molecular metabolism, adipocyte biology in particular, will provide the applicant scientific and career advices. The overall goal of the project is to study molecular phenotype of the newly isolated beige fat cells and delineate mechanisms responsible for beige cell commitment and regulation. Obesity is associated with diabetes, hypertension, dyslipidemia and cardiovascular disease. Obesity is essentially a disorder of energy balance, in which intake exceeds expenditure. There are two major types of adipose tissues in mammals, white and brown. White adipose tissue (WAT) stores excess energy in the form of triglycerides, whereas brown adipose tissue (BAT) can counteract obesity by safely burning off chemical energy through adaptive thermogenesis. In addition to classical brown adipose depots, UCP1 positive, brown fat-like cells have also been observed interspersed in WAT in response to cold exposure. Myf5-expressing embryonic progenitors give rise to the classic depots of BAT, but the brown-like cells in WAT (so-called beige cells) do not arise from this cell lineage. The cellular and developmental origin of beige cells remains unknown at present. Recent studies that revealed adult humans have functional BAT have raised the question regarding the molecular identity of these metabolically active cells. This proposal attempts to clarify the basic mechanisms regarding the control of beige cell development and activation, which will enable molecular definition of human brown fat and may suggest new approaches for the prevention and treatment of obesity and associated medical conditions. We clonally derived multiple lines of immortalized preadipocytes from inguinal WAT, interscapular BAT and epididymal WAT depots. Preliminary analysis shown that a subset of inguinal lines have a gene expression pattern similar to the brown lines and comparable Ucp1 expression in response to cAMP stimulations. These data strongly support our hypothesis that there is a distinct pool of progenitors in the inguinal depot that gives rise to the beige cells. Aim #1 is to compare physiological functions of beige fat cells, adaptive thermogenesis potential in particular, to thos of the bona fide brown fat cells. Mitochondrial content and respiration rate will be tested in cultured beige cells with brown cells as controls. Thermogenic gene expression will be assayed in transplanted fat pads derived by beige cells in immunodeficient mice. A role for beige fat cells in raising energy expenditure and protecting mice against obesity will be tested. Aim #2 is to determine molecular mechanisms by which beige fat is committed and regulated. A list of beige cell enriched beige fat gene signature will be identified through computational biology approach and validated in multiple in vitro and in vivo systems. Transcription factors as beige cell regulators will be identified through three complementary approaches and their functions will be tested in vitro via retroviral gain/loss of function in our immortalized cells and in vivo in transgenic mouse models. Aim #3 is to apply insights gained from this study to design novel strategies of prevention and treatment of obesity. To define molecular identify of human BAT, the expression levels of beige fat gene signature and brown fat gene signature will be investigated in PET positive human brown fat tissues. We will test and validate beige fat specific surface markers identified in aim 2 and design beige cell sorting strategies to purify beige cell populations from rodent and human adipose tissues. The Spiegelman laboratory and Harvard Medical School Longwood research community provide an ideal setting for training future independent investigators. This project will also bring together leading laboratories of the advisory committee that complement each other's expertise. These outstanding resources will maximize the potential for the applicant to successfully transition to an independent investigator. PUBLIC HEALTH RELEVANCE: We have identified and isolated the third type of fat cells - beige fat cells. This new type of fat cells regulate adaptive thermogenesis upon stimulation, which converts triglycerides into heat, therefore may play an important role in modulating metabolic homeostasis and counteracting obesity. This proposed study is aimed at analyzing molecular regulation and physiological function of beige fat cells both in mice and in human. The genes and pathways identified in this research project will be of wide interest to a number of scientists and pharmaceutical companies exploring various aspects of therapeutics of metabolic syndrome.